High-performance thin-layer chromatography plate blotting for liquid microjunction surface sampling probe mass spectrometric analysis of analytes separated on a wettable phase plate.

A blotting method that transfers analytes separated on wettable high-performance thin-layer chromatography (HPTLC) plates to a hydrophobic reversed-phase C8 HPLTC plate suitable for analysis with a liquid microjunction surface sampling probe electrospray ionization mass spectrometry system was described and demonstrated. The simple blotting procedure transfers the analyte from the wettable plate to the topmost surface of a rigidly backed, easy-to-mount hydrophobic substrate that already has been proven viable for analysis by this sampling probe/mass spectrometry system. The utility of the approach was demonstrated by the analysis of a four-component peptide mixture originally separated on a ProteoChrom® HPTLC cellulose sheet and then blotted onto the reversed-phase HPTLC plate.

Hydrophobic treatment enabling analysis of wettable surfaces using a liquid microjunction surface sampling probe/electrospray ionization-mass spectrometry system.

An aerosol application procedure involving one or more commercially available silicone-based products was developed to create hydrophobic surfaces that enable analysis of otherwise wettable, absorbent surfaces using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system. The treatment process resulted in a hydrophobic surface that enabled formation of the requisite probe-to-surface liquid microjunction for sampling and allowed efficient extraction of the analytes from the surface, but did not contribute significant chemical background in the mass spectra. The utility of this treatment process was demonstrated with the treatment of wettable high-performance thin layer chromatography plates, post-plate development, and their subsequent analysis with the sampling probe. The surface treatment process for different surface types was described and explained and the effectiveness of the treatment and subsequent analysis was illustrated using alkaloids from goldenseal (Hydrastis canadensis) root separated on a normal phase silica gel 60 F(254S) plate and peptides from protein tryptic digests separated on a ProteoChrom HPTLC Silica gel 60 F(254S) plate and a ProteoChrom HPTLC Cellulose sheet. This simple surface treatment process significantly expands the analytical surfaces that can be analyzed with the liquid microjunction surface sampling probe, and therefore, also expands the analytical utility of this liquid extraction based surface sampling approach.

Direct sampling and analysis from solid-phase extraction cards using an automated liquid extraction surface analysis nanoelectrospray mass spectrometry system.

Direct liquid extraction based surface sampling, a technique previously demonstrated with continuous flow and autonomous pipette liquid microjunction surface sampling probes, has recently been implemented as a liquid extraction surface analysis (LESA) mode on a commercially available chip-based infusion nanoelectrospray ionization (nanoESI) system. In the present paper, the LESA mode was applied to the analysis of 96-well format custom-made solid-phase extraction (SPE) cards, with each well consisting of either a 1 or a 2 mm diameter monolithic hydrophobic stationary phase. These substrate wells were conditioned, loaded with either single or multi-component aqueous mixtures, and read out using the commercial nanoESI system coupled to a hybrid triple quadrupole/linear ion trap mass spectrometer or a linear ion trap mass spectrometer. The extraction conditions, including extraction/nanoESI solvent composition, volume, and dwell times, were optimized in the analysis of targeted compounds. Limit of detection and quantitation as well as analysis reproducibility figures of merit were measured. Calibration data was obtained for propranolol using a deuterated internal standard which demonstrated linearity and reproducibility. A 10× increase in signal and cleanup of micromolar angiotensin II from a concentrated salt solution was demonstrated. In addition, a multicomponent herbicide mixture at ppb concentration levels was analyzed using MS(3) spectra for compound identification in the presence of isobaric interferences.

Characterizing the Impact of Spray Dried Particle Morphology on Tablet Dissolution Using Quantitative X-Ray Microscopy.

For oral solid dosage forms, disintegration and dissolution properties are closely related to the powders and particles used in their formulation. However, there remains a strong need to characterize the impact of particle structures on tablet compaction and performance. Three-dimensional non-invasive tomographic imaging plays an increasingly essential role in the characterization of drug substances, drug product intermediates, and drug products. It can reveal information hidden at the micro-scale which traditional characterization approaches fail to divulge due to a lack of resolution. In this study, two batches of spray-dried particles (SDP) and two corresponding tablets of an amorphous product, merestinib (LY2801653), were analyzed with 3D X-Ray Microscopy. Artificial intelligence-based image analytics were used to quantify physical properties, which were then correlated with dissolution behavior. The correlation derived from the image-based characterization was validated with conventional laboratory physical property measurements. Quantitative insights obtained from image-analysis including porosity, pore size distribution, surface area and pore connectivity helped to explain the differences in dissolution behavior between the two tablets, with root causes traceable to the microstructure differences in their corresponding SDPs.

Direct analysis of reversed-phase high-performance thin layer chromatography separated tryptic protein digests using a liquid microjunction surface sampling probe/electrospray ionization mass spectrometry system.

The sampling, ionization and detection of tryptic peptides separated in one-dimension on reversed-phase high-performance thin layer chromatography (HPTLC) plates was performed using liquid microjunction surface sampling probe electrospray ionization mass spectrometry. Tryptic digests of five proteins [cytochrome c, myoglobin, beta-casein, lysozyme and bovine serum albumin (BSA)] were spotted on reversed phase HPTLC RP-8 F254s and HPTLC RP-18 F254s plates. The plates were then developed using 70/30 methanol/water with 0.1M ammonium acetate. A dual purpose extraction/electrospray solution containing 70/30/0.1 water/methanol/formic acid was infused through the sampling probe during analysis of the developed lanes. Both full scan mass spectra and data dependent tandem mass spectra were acquired for each development lane to detect and verify the peptide distributions. Data dependent tandem mass spectra provided both protein identification and sequence coverage information. Highest sequence coverages were achieved for cytochrome c and myoglobin (62.5% and 58.3%, respectively) on reversed phase RP-8 plates. While the tryptic peptides were separated enough for identification, the peptide bands did show some overlap with most peptides located in the lower half of the development lane. Proteins whose peptides were more separated gave higher sequence coverage. Larger proteins such as beta-casein and BSA which were spotted in lower relative amounts gave much lower sequence coverage than the smaller proteins.

Extraction, separation, and fluorometric analysis of selected environmental contaminants.

Teratogenic, carcinogenic, and pervasive endocrine disrupting compounds (EDCs) in suspect water systems present an immediate threat to both the environment and potable water supplies. The US Environmental Protection Agency mandated research regarding suspect EDCs, personal care products, and pesticide pollution requires the use of suitable methods of analysis that can perform extraordinarily well in the field and show low "cost to benefit" ratios. Such methods must increasingly address the need for enhanced sensitivity and selectivity in interrogating complex mixtures. Here, several device and method optimization strategies, specific for these classes of compounds, are discussed and include, among others, the preconcentration of field samples utilizing high performance extraction disk cartridges. Matrix interferences and undetectable concentration levels are eliminated by the way of an RP extraction technique that is vital to obtaining detectable concentrations of target analytes in the ppb range. Established capillary EKC methods are modified and implemented on a short capillary for low nanoliter discrete injections, efficient separations, and detection with LIF of analyte mixtures. Efficient separations were achieved with plate counts ranging from 10(3) to 10(4). Analytes spiked into real sample matrices at ppb levels were easily separated and detected via LIF using a He/Cd laser operating at 325 nm. The presented techniques are valuable in determining both the presence and concentration of suspect contaminates in different water systems. The methods presented here could easily be extended to microfluidic platforms with little to no optimization for on-site testing.

Dual function surface-enhanced Raman active extractor for the detection of environmental contaminants.

Surface-enhanced Raman spectroscopy (SERS) has lagged behind other analytical techniques utilized in routine chemical analyses despite the information-rich spectra produced. This can be attributed in part to the difficulties in finding effective substrates that provide high sensitivity and highly reproducible SERS responses. Herein, we have developed a silver-coated polypropylene filter (AgPPF) as a highly sensitive and promising SERS substrate for the detection of environmentally significant chemicals, including selected pharmaceuticals, personal care products, and possible endocrine disruptors. The present approach involves preconcentration studies of selected environmental compounds with 3M's High Performance Extraction Disc Cartridges (HPEDCs) and characterization of the same HPEDC filters as a SERS substrate. The polypropylene microfiber prefilter that encloses the empore extraction medium is modified by physical vapor deposition with silver and used as the SERS substrate. The HPEDC itself is used to concentrate analytes into a desired concentration range, achieving maximum sensitivity. Surface roughness and nanoscale structure of silver films on the microfiber were characterized by atomic force microscopy (AFM). The mass thickness of the silver and volume were optimized for the highest SERS signal using rhodamine 6G as a model compound. A novel component of our HPEDC studies involves the hyphenation of the concentration protocol and SERS. The hyphenation of sample concentration and SERS allows more sensitive surface-enhanced detection. Mitoxanthrone dihydrochloride, crystal violet dye, 4-aminobenzoic acid, and rhodamine 6G were employed to study the sensitive SERS detection and were observed as low as 5 x 10(-8) M to 1 x 10(-10) M without any preconcentration step. Additionally, SERS signatures of some flavonoids, targeted as possible Endocrine Disrupting Chemicals by the US Environmental Protection Agency, such as Apigenin and Daidzein, are reported along with their sensitive detection down to ng/mL for the first time with preconcentration.

Correlation between peak capacity and protein sequence coverage in proteomics analysis by liquid chromatography-mass spectrometry/mass spectrometry.

Liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) allows the acquisition of a vast amount of analytical data in the aim of identifying peptides and proteins. Difficulties arise when attempting to identify proteins from the results of analyses of their peptide digests. We investigated possible quantitative correlations between the peak capacity achieved in chromatographic analyses and the protein sequence coverage. For this purpose, we used high performance liquid chromatography (HPLC) columns packed with fully and superficially porous (shell) particles of average sizes ranging from 2.6 to 15 microm in diameter. We found that the sequence coverage of bovine serum albumin obtained with a 10-min gradient elution run on a column packed with 2.6 microm shell particles is greater than that provided by 40-min gradients run on columns packed with 15 or 10 microm particles and equal to those yielded by the same 40-min gradient run on columns packed with 3 and 5 microm particles.